Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the present invention is related to the fields of medicine and medical treatment, specifically to the field of ultrasound treatment, and more particularly to an enhancement agent for HIFU treatment, which can increase acoustic energy deposition at the target location during HIFU treatment, and a method for screening the enhancement agents for HIFU treatment.
• High-intensity focused ultrasound as a new technique to treat tumors and other diseases has already been recognized in clinical applications.
• HIFU employs focused ultrasound, which provides continuous, high-intensity ultrasound energy at the focus, resulting in instantaneous thermal effects (65-100 °C), cavitation effects, mechanical effects and sonochemical effects, to selectively cause coagulative necrosis at the focus, and prevent tumors from proliferation, invasion and metastasis.
• the transmitting power of the therapeutic transducer might be increased in order to improve the therapeutic efficiency, the normal tissue along the pathway of the ultrasound transmission is more likely to be burned.
• the ribs are usually removed in order to increase the energy deposition at the target location, shorten the treatment time and improve therapeutic effects.
• the noninvasiveness of HIFU treatment cannot be ensured, which is undesirable for the patients and doctors.
• One objective of the present invention is to provide an enhancement agent for high intensity focused ultrasound (HIFU) treatment, which can enhance the acoustic energy deposition at target location during HIFU treatment.
• HIFU high intensity focused ultrasound
• Another objective of the present invention is to provide a method for screening the enhancement agent for HIFU treatment.
• a further objective of the present invention is to provide use of an enhancement agent for HIFU treatment to increase the effectiveness of HIFU treatment.
• the present invention provides an enhancement agent for HIFU treatment
• the enhancement agent is a substance that can enhance acoustic energy absorption at the target location to be treated with HIFU after its administration to a biological body, i.e. a substance that can be used to reduce the acoustic energy needed to cause lesions of a target tissue (tumor and non-tumor tissue) per unit volume of the tissue during HIFU treatment.
• the types of the substances used as the enhancement agents for HIFU treatment are not particularly limited, as long as the substances can change the acoustic environment of the target tissue and promote therapeutic acoustic energy absorption and deposition at the target tissue to effectively decrease the energy efficiency factor (EEF) of the target tissue. Therefore, the enhancement agents for HIFU treatment in the present invention can be solid, liquid or gas.
• the term "lesion” refers to the substantial change in the physiological state of a tumor tissue, generally refers to the coagulative necrosis of a tumor tissue.
• Energy efficiency factor EEF
• EEF Energy efficiency factor
• a substance that greatly decreases the EEF of the target tissue after its administration is more suitable to be used as the enhancement agent for HIFU treatment.
• the enhancement agent for HIFU treatment decreases the EEF of the target tissue after its administration.
• the ratio between the EEF of the target tissue measured before the administration of the enhancement agent (i.e. EEF (base) ) and the EEF of the target tissue measured after the administration of the enhancement agent (i.e. EEF (measurement) ) is more than 1, preferably more than 2, and more preferably over 4.
• the upper limit of the ratio is not particularly limited and a higher ratio is preferred.
• the enhancement agent for HIFU treatment is a biocompatible material with a particle size ranging from 10nm-8 ⁇ m, which can be administered via intravenous, arterial, or topical injections and can decrease the EEF of the target tissue after its administration.
• the ratio between the EEF of the target tissue before administration of the enhancement agent (i.e. EEF (base) ) and the EEF of the target tissue after administration of the enhancement agent (EEF (measurement) ) is more than 1, preferably more than 2, and more preferably over 4.
• the upper limit of the ratio is not particularly limited and a higher ratio is preferred.
• the enhancement agents for HIFU treatment of the present invention may be encapsulated by a lipid membrane, protein membrane or saccharide membrane, or may be in a naked form without being encapsulated.
• the enhancement agent for HIFU treatment may be encapsulated by a lipid membrane in order to improve the target specificity of the enhancement agent.
• the enhancement agent for HIFU treatment can be administered in a naked form without being encapsulated in the lipid membrane, protein membrane or saccharide membrane, as long as no blood vessel blockage would be induced when the enhancement agent for HIFU treatment is administered intravenously.
• the enhancement agent for HIFU treatment in order to make the enhancement agent for HIFU treatment according to the present invention target a specific tumor tissue, for example, hepatic tumor, kidney tumor, bone tumor, breast cancer and uterine fibroids, substances having a specific affinity to the tumor tissue or the focus, such as a tumor-specific antibody, may be added to the enhancement agent for HIFU treatment.
• a specific tumor tissue for example, hepatic tumor, kidney tumor, bone tumor, breast cancer and uterine fibroids
• substances having a specific affinity to the tumor tissue or the focus such as a tumor-specific antibody
• the enhancement agent for HIFU treatment comprises a discontinuous phase comprised of a core encapsulated by a membrane-forming material, and a continuous phase comprised of aqueous medium.
• the discontinuous phase is uniformly dispersed in the continuous phase, and the particle size of the discontinuous phase ranges from 10nm to 8 ⁇ m;
• the membrane-forming material is biocompatible and the core consists of gas, liquid or nanometer-sized biocompatible solid.
• Such enhancement agent for HIFU treatment is suitable to be administered intravenously.
• the enhancement agent for HIFU treatment that consists of a biocompatible gas encapsulated by a membrane-forming material
• a microbubble enhancement agent the enhancement agent for HIFU treatment that consists of the liquid encapsulated by a membrane-forming material
• a particle enhancement agent wherein the liquid is classified into two categories: liquid which does not undergo a liquid-gas phase transition at 38-100°C, and liquid which undergoes a liquid-gas phase transition at 38-100°C (specifically, the liquid will turn into gas within an animal body or human body during HIFU treatment);
• the enhancement agent for HIFU treatment that consists of nanometer-sized biocompatible solid encapsulated by a membrane-forming material, is hereinafter referred to as a "plasmid" enhancement agent.
• the amount of the membrane-forming material contained in the enhancement agent is 0.1-100g/L, preferably 0.5-50g/L, and more preferably 0.5-20g/L.
• the membrane-forming material includes: lipids, such as 3-sn-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol sodium salt, 1,2-distearoyl-sn-glycero-3-phosphatidylcholine, sodium 1,2-dipalmitoyl-sn-glycero-3-phosphatidate, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, phosphatidylserine and hydrogenated phosphatidylserine, cholesterol, and glycolipide; saccharides, including, for example, glucose, fructose, sucrose, starch and the degradation products thereof; proteins, such as albumin, globulin, fibrinogen, fibrin, hemoglobin
• the amount of the gas contained in the enhancement agent is 5-200ml/L, preferably 20-150ml/L, and more preferably 20-100ml/L.
• the gas includes, for example, air, nitrogen, carbon dioxide, fluorohydrocarbon gas, such as perfluoroethane, perfluoropropane, perfluorobutane, alkane gases, such as butane, cyclobutane, pentane, hexane, sulfur hexafluoride, and the like.
• microbubble ultrasound contrast agents widely used in ultrasound imaging may be used as the enhancement agent for HIFU treatment of the present invention.
• the present invention provides use of microbubble ultrasound contrast agents as the enhancement agent for HIFU treatment of the present invention.
• the amount of the liquid contained in the enhancement agent is 5-200g/L, preferably 10-100g/L, and more preferably 20-80g/L; if liquid that undergoes a liquid-gas phase transition at 38-100°C, for example, C 5 -C 6 alkanes such as n-pentane, i-pentane and the like., and C 5 -C 12 fluorohydrocarbons such as perfluoropentane, dihydrodecafluoropentane and the like, is used, the amount of the liquid contained in the enhancement agent is 5-200ml/L, preferably 10-100ml/L, and more preferably 20-80ml/L.
• a fat emulsion for injection is an aqueous emulsion of fat, consists of refined soybean oil encapsulated by a phospholipin membrane, which is dispersed in water, and is suitable for intravenous injection.
• this kind of emulsion is commercially available, including, but not limited to, Intralipos® (fat emulsion injection), OMNILIPID® (fat emulsion injection), and "fat emulsion (long chain)” or “fat emulsion (medium chain triglycerides/long chain triglycerides)” listed in the catalogue of basic medicines of the state (China).
• These fat emulsions can be used as the enhancement agents for HIFU treatment of the present invention.
• the present invention provides use of fat emulsions as the enhancement agents for HIFU treatment of the present invention.
• the nanometer-sized biocompatible solid When using a plasmid enhancement agent, the nanometer-sized biocompatible solid includes nanometer-sized magnetic biomaterials such as superparamagnetic iron oxide (SPIO), nanometer-sized hydroxylapatite (HAP), nanometer-sized calcium carbonate and the like.
• the nanometer-sized biocompatible solid has a particle size ranging from 1nm to 500nm, preferably from 1nm to 200nm, and more preferably from 10nm to 100nm.
• nanometer-sized biocompatible solids as mentioned above can be used as the enhancement agents of the present invention by themselves. Therefore, the present invention provides use of nanometer-sized biocompatible solids as the enhancement agent of the present invention.
• the enhancement agent may contain an emulsifier.
• the emulsifier is typically selected from the group consisting of ethylene glycol mono-C 16-18 -fatty acid esters, diethylene glycol mono-C 16-18 -fatty acid esters, diethylene glycol di-C 16-18 -fatty acid esters, triethylene glycol mono-C 16-18 -fatty acid esters, sorbitan fatty acid ester (Span type) emulsifiers, polysorbate (Tween type) emulsifiers, polyethylene glycol monolaurate-based emulsifiers, polyoxyethylene laurate-based emulsifiers, 3-sn-phosphatidylcholine (lecithin), cholic acid and the like.
• the amount of the emulsifier in the enhancement agent is 5-150g/L.
• the enhancement agent may also contain a stabilizing agent, such as carboxymethylcellulose sodium (CMC-Na), glycerin and the like.
• CMC-Na carboxymethylcellulose sodium
• the amount of the carboxymethylcellulose sodium contained in the enhancement agent may be 0.01-10g/L, preferably 0.05-0.6g/L, and more preferably 0.1-0.3g/L.
• the amount of the glycerin contained in the enhancement agent may be 5-100g/L.
• an inorganic or organic acid or base may be used to adjust the pH value of the enhancement agent in order to increase the stability of the enhancement agent.
• the particle enhancement agent may be adjusted to pH 7.0-9.0, preferably 7.5-8.5.
• the plasmid enhancement agent may be adjusted to pH 3.0-6.5, preferably 5.0-6.0.
• the methods for the preparation of the enhancement agent for HIFU treatment of the present invention are not particularly limited.
• the membrane-forming material, the gas, liquid or solid to be encapsulated, the emulsifier, the stabilizing agent and the like are sufficiently mixed and emulsified.
• the particle fat emulsion can be prepared according to the disclosures of Chinese Patent Application No. 97182319.7 (Entitled: Fat emulsion containing reducing sugar and method of sterilization) or Chinese Patent Application No. 02112860.X (Entitled: Fat emulsion for injection and method for producing the same).
• microbubble fluoro-carbon emulsion can be prepared according to the disclosures of Chinese Patent Application No. 96106566.4 (Entitled: Dextrose anhydride albumin ultrasound contrast agent containing perfluorohydrocarbons and method for producing the same), Chinese Patent Application No. 98119011.1 (Entitled: A ultrasound contrast agent for ultrasound diagnosis and method for producing the same) or Chinese Patent Application No. ZL 89100726.1 (Entitled: Method for the preparation of the particle used for ultrasound contrast and the ultrasound contrast agent).
• the membrane-forming material of the enhancement agent for HIFU treatment according to the present invention is preferably a biocompatible and degradable biomaterial, such as lipid, such that the enhancement agent can be injected intravenously, transported through the blood circulation smoothly, and then phagocytosed quickly by the tissues of the human body, which are full of reticuloendothelial cells. Therefore, a mass of enhancement agent can be deposited in the tissues of the human body in a certain time, thereby, the ultrasound absorption capacity of the tissue can be significantly enhanced, and the acoustic energy deposition at the target tissue during HIFU treatment can be increased and eventually the effectiveness of clinical HIFU treatment to ablate tumor cells can be improved greatly.
• the present invention is further directed to a method for increasing the energy deposition at the target location during the HIFU treatment, wherein, the method comprises administering an effective dosage of the enhancement agent of the present invention intravenously via continuous and rapid IV instillation or bolus injection to a patient at 0-168h before applying HIFU treatment to a patient.
• the effective dosage mentioned above varies with the type of tumor, weight of patient, location of tumor, volume of tumor and the like. However, a doctor or a pharmacist can easily determine the suitable dosage for different patients. For example, when using a microbubble enhancement agent, the dosage can be selected from the range of 0.005-0.1ml/kg, preferably 0.01-0.05ml/kg.
• the dosage when using a particle enhancement agent, if liquid that does not undergo a liquid-gas phase transition at 38-100°C is used, the dosage can be selected from the range of 0.01-5ml/kg, preferably 0.01-2.5ml/kg; and if liquid that undergoes a liquid-gas phase transition at 38-100°C is used, the dosage can be selected from the range of 0.005-0.1ml/kg, preferably 0.01-0.05ml/kg. When using a plasmid enhancement agent, the dosage can be selected from the range of 0.1-10ml/kg, preferably 0.1-5ml/kg.
• the present invention is also directed to a method for screening the enhancement agent for HIFU treatment, the method comprising:
• the present invention provides a treatment method for diseases, comprising administering the enhancement agent for HIFU treatment to a patient before applying the HIFU treatment to improve the therapeutic acoustic energy absorption capacity of the target tissue to be treated with HIFU.
• Example 1-1 Using liquid that does not undergo a liquid-gas phase transition at 38-100 °C for encapsulation.
• the following materials were mixed: 4g iodized oil for injection (purchased from Shanghai Chemical Reagent Company), 0.6g yolk lecithin for injection (purchased from Shanghai Chemical Reagent Company) and 1.25g glycerin for injection (purchased from Shanghai Chemical Reagent Company), and this mixture was dissolved and formed an oil phase after heating to 70°C. Distilled water containing 1% (w/v) F-68 emulsifier (purchased from Sigma Company) was added to the oil phase to a final volume of 17.5ml. The mixture was agitated to obtain a coarse emulsion. The coarse emulsion, which was poured into a boiling tube, was emulsified by sonication at a power of 350W for 2 minutes.
• the resulting uniformly emulsified iodized oil was sterilized through flowing steam at 100°C for 30 minutes.
• the final product had a pH of 7.5-8.5, iodine content of 0.13g/ml, particle size of less than 1 ⁇ m and osmotic pressure of 350mosm/kg H 2 O.
• Examples I-1-2 to I-1-4 were prepared according to the same method and procedures described in Example I-1-1 except that the iodized oil for injection was replaced with the soybean oil for injection as the core material, and the yolk lecithin for injection was replaced with the lecithin as the membrane-forming material.
• the particle enhancement agents for HIFU treatment of the present invention were obtained according to the formulation set forth below in Table 1.
• the enhancement agents were obtained as white emulsion liquids, which can be administered to animals and human beings via intravenous injection. The parameters of the products are also shown in Table 1.
• Example I-1-2 Example I-1-3
• Example I-1-4 Concentration of Soybean oil for injection in the enhancement agent (w/v) 10% 20% 10% Amount of Soybean oil for injection 100g 200g 100g Amount of Lecithin for injection 12g 12g 12g Amount of Glycerin for injection 22g 22g 16.7g
• Final volume after Water for injection added 1000ml 1000ml 1000ml pH (c.a.) 8 8 8 Particle size of the discontinuous phase 0.1-2 ⁇ m 1-5 ⁇ m 0.5-2 ⁇ m Osmotic pressure (mosm/kg H 2 O) 300 350 310 Energy MJ (kcal) 4.6 (1100) 8.4 (2000) 12.6 (3000)
• Example 1-2 Using liquid that undergoes a liquid-gas phase transition at 38-100°C for encapsulation.
• the following materials were mixed to a final volume of 1000ml: 3% (w/v) emulsifier Pluronic F-68 (purchased from Sigma Company), 0.5% (w/v) yolk lecithin (purchased from Shanghai Chemical Reagent Company), 5% (v/v) perfluoropentane (purchased from Sigma Company), and distilled water.
• the mixture was incubated on ice, sheared, and dispersed at 10000 rpm for 5 minutes to obtain a coarse emulsion.
• the coarse emulsion was emulsified in a high-pressure homogenizer at 4°C for two times.
• the resulting emulsion with a particle size of less than 1 ⁇ m was obtained by filtering through a 1 ⁇ m membrane filter.
• the final emulsion was divided and put into 15ml vials, and then was radiated by C 060 at 20KGY for 10 hours.
• the emulsion had a particle concentration of 10 9 /ml and was ref
• the following materials were mixed to a final volume of 1000ml: 6% (w/v) emulsifier Pluronic F-68 (purchased from Sigma Company), 1% (w/v) yolk lecithin (purchased from Shanghai Chemical Reagent Company), 10% (v/v) perfluoropentane (purchased from Sigma Company), and physiological saline solution.
• the mixture was incubated on ice, sheared, and dispersed at 10000 rpm for 5 minutes to obtain a coarse emulsion.
• the coarse emulsion was emulsified in a high-pressure homogenizer at 4°C for two times.
• the resulting emulsion with a particle size of less than 1 ⁇ m was obtained by filtering through a 1 ⁇ m membrane filter.
• the final emulsion was divided and put into 15ml vials, and then was radiated by Co 60 at 20KGY for 10 hours.
• the emulsion had a particle concentration of 10 9 /ml and was ref
• Fluoro-carbon emulsion enhancement agents for HIFU treatment of the present invention were prepared according to the same method and procedures described in Example I-2-1 with the materials and the amounts thereof set forth in Table 2. The parameters of the products are shown in Table 2. Table 2 Example I-2-3 Example I-2-4 Example I-2-5 Example I-2-6 Core material 2% (v/v) Perfluoropentane 5% (v/v) Perfluorohexane 10% (v/v) Perfluorohexane 10% (v/v) Dihydrodecafluoropentane Lecithin 1% (w/v) 2% (w/v) 2% (w/v) 2% (w/v) 2% (w/v) Glycerin 1% (w/v) 1 %(w/v) 1% (w/v) 1% (w/v) Pluronic F-68 5% (w/v) 3% (w/v) 5% (w/v) 5% (w/v) Final volume after distilled water added 1000ml 1000
• the following materials were mixed: 2.5g HAP with a particle size ranging from 1nm to 100nm (purchased from the Engineering Research Center for Biomaterials of Sichuan University), 0.3g yolk lecithin for injection (purchased from Shanghai Chemical Reagent Company) and 0.3g CMC-Na (purchased from Shanghai Chemical Reagent Company), and distilled water to a final volume of 100ml. After being uniformly mixed, the mixture was pH-adjusted with acetic acid to pH 5.0. The mixture was sonicated for 2 minutes at a power of 400W with the transmitter of the sonicator positioned 1.5 cm below the surface of the mixture. After sonication, a milk-white, uniformly dispersed, stable suspension was obtained.
• the particle size of the discontinuous phase of the resulting enhancement agent ranged from 10nm to 1000nm, mainly ranged from 100nm to 500nm.
• the following materials were mixed: 2.5g HAP with a particle size ranging from 1 nm to 100nm (purchased from the Engineering Research Center for Biomaterials of Sichuan University), 0.3g yolk lecithin for injection (purchased from Shanghai Chemical Reagent Company) and 1ml glycerin for injection, and distilled water to a final volume of 100ml. After being uniformly mixed, the mixture was pH-adjusted with acetic acid to pH 5.0. The mixture was sonicated for 2 minutes at a power of 400W with the transmitter of the sonicator positioned 1.5 cm below the surface of the mixture. After sonication, a milk-white, uniformly dispersed, stable suspension was obtained.
• the particle size of the discontinuous phase of the resulting enhancement agent ranged from 10nm to 1000nm, mainly ranged from 100nm to 500nm.
• Plasmid enhancement agents for HIFU treatment of the present invention were prepared according to the same method and procedures described in Example II-1 with the materials and the amounts thereof set forth in Table 3. The parameters of the products were are shown in Table 3. Table 3 Example II-3 Example II-4 Example II-5 Nnanometer-sized HAP (particle size) 25g/L (1-500nm) 25g/L (1-500nm) 50g/L (1-500nm) Lecithin 0.3g 0.3g 0.6g CMC-Na 0.3g 0.6g 0.3g Glycerin for injection 1ml 1ml 2ml Final volume after distilled water added 100ml 100ml 100ml PH (c.a.) 5.0 5.0 5.0 Particle size of the discontinuous phase 10-1000nm 10-1000nm 10-1000nm 10-1000nm Osmotic pressure (mosm/ kg. H 2 O) 275 (Isosmotic) 275 (Isosmotic) 275 (Isosmotic) 275 (Isosm
• HAP hydroxylapatite
• the New Zealand white rabbits were anaesthetized through intramuscular injection, fastened to the treatment bed of a High-intensity Focused Ultrasound Tumor Therapeutic System Model-JC manufactured by Chongqing Haifu (HIFU) Technology Co. Ltd., and then treated by using this System.
• the High-intensity Focused Ultrasound Tumor Therapeutic System Model-JC was composed of an adjustable power generator, a B-mode ultrasound monitoring system, a therapeutic transducer, a mechanical motion control system, a treatment bed and an acoustic coupling device.
• the therapeutic transducer of the System with a working frequency of 1MHz, diameter of 150mm, and focal distance of 150mm, using standard circulating degassed water with a gas content of no more than 3ppm, can produce a focal region of 2.3 ⁇ 2.4 ⁇ 26mm and deliver an average acoustic intensity of 5500W/cm 2 .
• the rabbit livers were pre-scanned by the B-mode scanner of the HIFU therapeutic system. Two slices with an interval of at least 2cm at an exposure depth of 2.0cm were measured.
• the left side of the rabbit liver (the left/middle lobe) was considered as the control lobe (which was administered with physiological saline solution), and the right side of the rabbit liver (the right lobe) was considered as the experimental lobe (which was administered with the enhancement agent for HIFU treatment as prepared in Example I-1-3, and also called the enhancement agent side).
• the control lobe and experimental lobe were reversely positioned in Group B.
• the exposure depth of HIFU treatment (i.e., the distance from the skin surface to the focal point) was also 2.0cm.
• the physiological saline solution was delivered via rabbit ear border vein at 50-60 drops/min. After 20 minutes, the left side of the rabbit liver (Group A) or the right side of the rabbit liver (Group B) was exposed to HIFU under single pulse exposure or multi-pulse exposure (line length: 1 cm, scanning speed: 3mm/s), and the gray scale changes and the time for exposure in target location were recorded. Then the focal point of the HIFU therapeutic system was moved over to the opposite site.
• the enhancement agent for HIFU treatment as prepared in Example I-1-3 was administered intravenously, the injection speed and the time being the same as that of the control lobe of liver. Then the right side of the rabbit liver (Group A) or the left side of the rabbit liver (Group B) was exposed to HIFU. The treatment modes used for both sides of the liver of the same rabbit were the same.
• the rabbits were sacrificed and dissected at 24 hours after HIFU treatment.
• the dimensions (length, width and thickness) of the coagulation necrosis zone of the rabbit liver lesions were measured.
• the EEF energy efficiency factor
• EEF ⁇ Pt / V , (J/mm 3 ).
• the EEFs were compared between and within Group A and Group B.
• P refers to the total acoustic power of a HIFU source (W);
• t refers to the total time of HIFU treatment (s);
• V refers to the volume of HIFU-induced lesions (mm 3 ).
• a substance that decreases the EEF of the target tissue after its administration is more suitable to be used as the enhancement agent for HIFU treatment according to the present invention. The results are shown in Table 4.
• a High-intensity Focused Ultrasound Tumor Therapeutic System Model-JC manufactured by Chongqing Haifu (HIFU) Technology Co. Ltd. was used to radiate the livers of the white rabbits in the experimental group and the control group under single pulse exposure.
• the power for exposure was 220W; the frequency was 1.0MHZ; the exposure depth was 20mm and the exposure was stopped when coagulative necrosis occurred.
• the measured data were expressed with mean value ⁇ SD, processed by the statistics software SPSS 10.0 for Windows using independent and paired sample test.
• the enumeration data were determined by using chi-square ( ⁇ 2 ) test.
• the comparisons between the EEF of the control group and the EEF of the experimental group are shown in Table 5.
• the HIFU gynaecological therapeutic apparatus CZF-1 is composed of a power source, an applicator and circulating water as described in Chinese Patent No. 01144259.X .
• the parameters in this test were set up as follows: power: 4.05W; frequency: 11MHz; and pulse: 1000Hz.
• the enhancement agent for HIFU treatment as prepared in Example I-1-3 was delivered via rabbit ear border vein to the rabbits in the experimental group at 50-60 drops/minute for 20 minutes and the physiological saline solution was delivered to those in the control group at 50-60 drops/minute for 20 minutes.
• the rabbit was fastened to a workbench in supine position.
• the laparotomy was carried out with a 4-5cm incision in the midsection and the rabbit liver in the abdominal cavity was exposed and pulled out slightly after the abdomen wall was opened layer by layer.
• One or two exposure spots on each liver lobe were introduced for each exposure duration of 3s, 6s, and 9s. The experiments were carried out using the parameters as mentioned above after the exposure spots were introduced. After the lesions were generated, the rabbit liver was put back to the abdominal cavity and the abdomen wall was sutured layer by layer.
• the High-intensity Focused Ultrasound Tumor Therapeutic System Model-JC is composed of an adjustable power generator, a B-mode ultrasound monitoring system, a therapeutic transducer, a mechanical motion control system, a treatment bed, and an acoustic coupling device.
• the therapeutic transducer of the System with working frequency of 1MHz, diameter of 150mm, and focal distance of 150mm, using standard circulating degassed water with gas content of less than or equal to 3ppm, can produce a focal region of 2.3 ⁇ 2.4 ⁇ 26mm and deliver an average acoustic intensity of 5500W/cm 2 .
• the transducer used in this study was 150mm in diameter, and it had a focal distance of 135mm, a working frequency of 1.0MHz and an acoustic power of 200W.
• the exposure depth was 20mm, and a discontinuous single pulse exposure with exposure duration of 3s and interval of 5s was applied.
• the physiological saline solution (0.02ml/kg) was quickly delivered via rabbit ear border vein to each rabbit, and the rabbit liver was exposed with HIFU using single pulse exposure 60 seconds later for the control side.
• the enhancement agent for HIFU treatment as prepared in Example I-2-1 (0.02ml/kg) was quickly delivered via rabbit ear border vein to each rabbit, and the other plane of the same rabbit liver of the control side was exposed to HIFU at 60 seconds later for the experimental side.
• the ultrasound exposures finished when gray-scale changes occurred at the target location. If there are no gray-scale change to be seen, the total exposure duration should be no more than 20s. Three days after ultrasound exposure, the rabbits were sacrificed by breaking their necks and were then dissected.
• the volume (V) of coagulative necrosis of rabbit liver was measured.
• the median of the EEFs was 6.0160 on the control side and 1.2505 on the experimental side.
• the results of this study show that the fluoro-carbon emulsion increases the effectiveness of HIFU to cause lesions of the rabbit livers.
• the mean of the EEF in the control side is 4.81 times as much as the mean of the EEF in the experimental side.
• the goats were shaved at the right bosom and the right abdomen on the day of the study.
• a High-intensity Focused Ultrasound Tumor Therapeutic System Model-JC manufactured by Chongqing Haifu (HIFU) Technology Co. Ltd. was used to radiate these yellow goats.
• the transducer used in this study was 150mm in diameter, had a focal distance of 135mm, a working frequency of 0.8MHz, and an acoustic power of 220W.
• the exposure depth was 30mm, and a discontinuous single pulse exposure with exposure duration of 3s and interval of 5s was applied. Ribs of all the goats were not removed.
• a pre-scan was carried out before HIFU exposure and the areas for exposure including 4 planes were selected. One exposure spot was introduced on each plane, and two-dimensional ultrasound was used to monitor rib clearance.
• the physiological saline solution (0.02ml/kg) was quickly delivered intravenously via ear border to each goat, and the goat liver was exposed to HIFU 60 seconds later, and two exposure spots were introduced on each goat on the control side.
• the enhancement agent for HIFU treatment as prepared in Example I-2-1 (0.02ml/kg) was quickly delivered intravenously via ear border to each goat, and the goat liver was exposed to HIFU 60 seconds later, and two exposure spots were introduced on each goat on the experimental side. When gray-scale changes occurred at the target location, the exposures were repeated another 4 or 5 times.
• the total exposure duration should be no more than 200s.
• the volume (V) of coagulative necrosis of goat liver was measured.
• a pre-scan was carried out before HIFU exposure and the areas for exposure including 1 plane on the upper pole of the kidney and 1 plane on the lower pole of the kidney respectively were selected. One exposure spot was introduced on each plane, and two-dimensional ultrasound was used for observation. The right ribs would be avoided if they become obstacles.
• the physiological saline solution (0.02ml/kg) was quickly delivered intravenously via ear border to each goat, and the goat kidney was exposed to HIFU under single pulse exposure 30 seconds later on the control side.
• the enhancement agent for HIFU treatment as prepared in Example I-2-1 (0.02ml/kg) was delivered quickly intravenously via ear border to each goat, and the goat kidney was exposed to HIFU 60 seconds later on the experimental side.
• the group that was exposed to HIFU at 24 hours after injection was called the first experimental group and the group that was exposed to HIFU 48 hours after injection was called the second experimental group.
• a High-intensity Focused Ultrasound Tumor Therapeutic System Model-JC manufactured by Chongqing Haifu (HIFU) Technology Co. Ltd. was used to radiate the livers of the rabbits in the control group and two experimental groups under single pulse exposure.
• the High-intensity Focused Ultrasound Tumor Therapeutic System Model-JC is composed of an adjustable power generator, a B-mode ultrasound monitoring system, a therapeutic transducer, a mechanical motion control system, a treatment bed, and an acoustic coupling device.
• the therapeutic transducer of the System with working frequency of 1MHz, diameter of 150mm, and focal distance of 150mm, using standard circulating degassed water with gas content of less than or equal to 3ppm, can produce a focal region of 2.3x2.4x26mm and deliver an average acoustic intensity of 5500W/cm 2 .
• the acoustic power for exposure was 220W
• the frequency was 1.0MHZ
• the exposure depth was 20mm
• the exposure duration was 15 seconds.
• the animals were sacrificed and dissected after HIFU exposure.
• the dimensions of coagulation necrosis at target location were measured.
• the EEFs needed to produce certain coagulative necrosis in the rabbit livers in the control group and two experimental groups are shown in Table 7.
• each rabbit in HAP groups was administered with HAP suspensions as prepared in Example III varying in concentrations by rapid injection ( ⁇ 5s) via rabbit ear border vein with a dosage of 2-3ml per 1kg body weight. Then they were flushed with 1ml physiological saline solution in order to ensure that the suspension had entered into the body completely.
• Each rabbit in the control group was administered with physiological saline solution (2ml/kg) by rapid injection via rabbit ear border vein.
• the rabbits were denuded with 8% sodium sulfide on the right bosom and abdomen.
• the rabbits were anesthetized by an intramuscular injection of Sumianxin (0.2ml/kg), prior to HIFU treatment, and the abdomen wall was incised under aseptic conditions to fully expose the liver.
• HIFU gynaecological therapeutic apparatus CZF-1 manufactured by Chongqing Haifu (HIFU) Technology Co. Ltd. was used to radiate the rabbit livers.
• the HIFU gynaecological therapeutic apparatus CZF-1 is composed of a power source, an applicator, and circulating water as disclosed in Chinese Patent No. 01144259.X .
• the parameters in this test were set up as follows: frequency: 9.85MHz, power: 5W, focal distance: 4mm, and treatment mode: single pulse exposure. Three exposure spots for one cycle and 2 or 3 exposure cycles for each liver were introduced. The exposure duration was 10 seconds.
• the incision was sutured after HIFU treatment. Twenty-four hours later, the rabbits were sacrificed by rapid injection of 10ml air via rabbit ear border vein. The dimensions of coagulation necrosis formed at target location were measured and the EEF was calculated.
• Table 8 Group Dosage n V/mm 3 EEF Control group 2ml/kg 30 95.3 ⁇ 21.6 0.39 ⁇ 0.09 HAP group 1 50mg/kg 30 153.1 ⁇ 41.8 0.24 ⁇ 0.05 HAP group 2 100mg/kg 25 223.2 ⁇ 55.1 0.19 ⁇ 0.01 HAP group 3 150mg/kg 21 287.7 ⁇ 47.9 0.13 ⁇ 0.00 Note: "n" in the table refers to the number of the exposure spots.
• the nanometer-sized HAP can greatly enhance the therapeutic effects of HIFU in vivo and that HIFU treatment was more effective when more HAP dosage was applied.
• mice Ten rabbits in the control group were administered with physiological saline solution (2ml/kg) and the rabbit livers were scanned with HIFU at 24 hours after the administration of physiological saline solution. Prior to the HIFU treatment, these rabbits were denuded with 8% sodium sulfide on the right bosom and abdomen. The rabbits were anesthetized by an intramuscular injection of Sumianxin(0.2ml/kg) and secured to a High-intensity Focused Ultrasound Tumor Therapeutic System Model JC-A.
• the High-intensity Focused Ultrasound Tumor Therapeutic System Model JC-A was manufactured by the Institute of Ultrasound Engineering in Medicine, Chongqing University of Medical Sciences, and the manufacture thereof was approved by the State Food and Drug Administration in China with the registration No. 99-301032.
• This system consists of a real time ultrasound monitoring and positioning apparatus and a therapeutic apparatus. Circulating degassed water was used as the acoustic coupling agent, which contained a gas of less than 3 ⁇ 10 -6 .
• Therapeutic parameters were set up as follows: power: 220W, frequency: 1MHz, focal distance: 150mm and focal length: 12mm.
• the therapeutic applicator could move in the directions of X, Y and Z-axis freely.
• each rabbit was immersed in the circulating degassed water and the rabbit liver was imaged clearly under B-mode ultrasound.
• One or two exposure spots could be introduced on each liver under single pulse exposure. Each exposure spot was introduced for a fixed exposure period of 15s with an exposure depth of 20mm.
• the rabbits were sacrificed by rapid injection of 10ml air via rabbit ear border vein at 24 hours after HIFU treatment, and the liver was exteriorized and incised along the acoustic pathway, showing the section of maximum coagulative necrosis area. Then the shape of the coagulative necrosis area was determined, and the dimensions of the coagulative necrosis area as determined by TTC-staining were measured. Then the EEF was calculated.
• the coagulative necrosis area formed in the HAP groups using nanometer-sized HAP was larger than that in the control group (p ⁇ 0.05); the EEF needed for HIFU treatment in the HAP groups decreased greatly in comparison with the control group. Also, in the HAP groups, the largest coagulative necrosis area was obtained with HIFU exposure at 24 hours and 48 hours after the HAP injection, and accordingly the least EEF was needed. It is probably most effective to carry out the HIFU exposure at 24 hours and 48 hours after the HAP injection. If the time to carry out the HIFU exposure after the HAP injection were postponed, a smaller necrosis area would be formed.
• the nanometer-sized HAP can greatly enhance the therapeutic effects of HIFU in vitro, and the HIFU treatment was more effective when the HIFU exposures were carried out at 48 to 72 hours after the HAP injection.
• the exposures were carried out under the power of 200W at frequency of 1.0MHz with exposure depth of 20mm for a certain exposure period. Three days later, the dimensions of lesions induced in rabbit livers were measured and the EEFs calculated. The measured data were expressed with mean value ⁇ SD, processed by the statistics software SPSS 10.0 for Windows using independent and paired sample test. The enumeration data was determined by using chi-square ( ⁇ 2 ) test. The results are shown in the following Table 10. Table 10 The EEFs of the control group and the experimental group Group EEF(J/mm 3 ) Control group 12.83 ⁇ 10.99 Experimental group 2.70 ⁇ 1.29* * P ⁇ 0.001 when compared to the control group.
• the enhancement agent for High-Intensity Focused Ultrasound (HIFU) treatment of the present invention can change the acoustic environment of the target location greatly and can reduce the acoustic energy needed to cause lesions of a target tissue (tumor and non-tumor tissue) per unit volume of the tissue during HIFU treatment. Accordingly, deep-seated and large-sized tumors can be treated with HIFU treatment more effectively under a certain acoustic power without damaging the normal tissues along the acoustic pathway. It becomes possible to use the enhancement agent for HIFU treatment of the present invention for applying HIFU treatment effectively to a patient with a hepatic tumor that is blocked by the ribs in therapeutic acoustic pathway without removal of the ribs.

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